- The female spermatheca is responsible for the maintenance, nourishment, and protection of the male sperm against damage during storage. - In situ RNA hybridization confirmed tissue-specific expression of the eight transcripts.. - aegypti is carried out across much of the tropics and subtropics [3]. - Though male- derived nutrients also transferred to the female during insect mating help nourish the sperm from a few hours to a few days, ultimately it is up to the female sperma- theca to maintain the viability of the male sperm [5, 6].. - 2020 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0. - Full list of author information is available at the end of the article. - The spermathecal gland is attached to the portion of the reservoir wall closer to the duct, while GC are individually attached to the duct. - Reservoir and duct GC secrete components into the lumens of the spermathecae through cuticle interruptions or pores.. - The spermathecal duct is externally covered by a muscu- lar layer, the spermathecal muscle, that is responsible for the contraction of the duct . - A general view of the morphology of Ae. - The characterization of molecules produced by the sperma- thecae and molecules directly associated with sperm via- bility can provide a further understanding of the function of these pivotal organs and may be used as tar- gets for novel control approaches.. - 1 Schematic representation of a section of the Ae. - Additional criteria for inclusion of the eight transcripts in our downstream studies included: 1) differential ex- pression levels between virgin and inseminated. - genome-wide coverage, and almost 22.7 million were lo- calized to the spermathecae of the inseminated females representing 77.57% of the Ae. - Expression levels of the spermathecal genes were sepa- rated from housekeeping genes by comparing the results from spermathecomes to whole body expression of fe- male and male (F-test with p-value of 0.05 after Bonfer- roni correction for multiple comparisons). - 1), where RPKM = 1 corresponds to the value of the constitutive expression found in the whole. - The transcripts identified in distinct clus- ters of the male and female differentially expressed genes (DEG) common to the spermathecomes, including genes overexpressed in the two spermathecomes, were grouped in a heat map graph representation (Fig. - To distinguish between the expression levels of the previously identified genes in the virgin or inseminated spermathecae, coding sequences were also compared among themselves and those whose expression differed by at least eight-fold were pre-selected (Additional file 1:. - A total of 661 DEG with at least an eight- fold increase over the expression levels of the house- keeping gene were identified, annotated, and divided into 21 functional classes (Additional file 1: Table S1).. - Of the 661 DEG identified, 111 were highly expressed (>. - The expression profile of each of the eight selected transcripts was assessed by RT-PCR in both the virgin and inseminated spermathecae, as well as the sper- mathecal content (i.e., the sperm within the reservoir lumen), and normalized to the expression levels of the. - Atro-1 was significantly downregu- lated in the inseminated compared with the virgin spermathecae (P and was undetected in the reservoir content of the inseminated. - The expression profiles of the eight selected transcripts were assessed separately for midgut, ovaries, and carcasses (i.e., the body without gut, ovaries, and spermathecae) of both virgin (sugar-fed only, non-vitellogenic ovaries) and inseminated females (sugar and blood-fed, with devel- oped/vitellogenic ovaries). - Additionally, no difference was detected regarding expression levels for the eight transcripts comparing ovaries before or after egg develop- ment of the inseminated females (and blood-fed). - Fitness parameters, including overall survival, blood feeding, fer- tility, and egg morphology, as well as effects on the spermatheca morphology were assessed as a result of the dsRNA injections and are discussed separately below. - A summary of the phenotypic effects provided by the KD effects for each target gene is shown in Table 1, and Additional file 1: Table S7.. - Table 1 Summary of the phenotypic effects observed after dsRNA injection for each target gene of spermatheca of Ae. - whether KD affected the spermathecae/sperm only, or whether non-spermathecal tissues of the reproductive system were also affected. - For dsGld-injected females, the area of the eggs laid was larger than the area of eggs laid by the control females (P <. - 0.0001), whereas for all the other treatments the area of the eggs laid was smaller than the control-laid eggs (P <. - To identify any effects of the dsRNA injections on the spermathecal morphology and sperm integrity, overall spermathecae and sperm morphologies (for sperm inside the spermathecae) were investigated. - The morphologies of the spermathecal duct, the glandular portion, and the reservoir were not altered by the injections. - Surrounding the internal part of the reservoir and continuously with the spermathecal duct, a well-structured thicker cuticle was observed (Additional file 3).. - A summary of the mea- surements of the dsRNA-injected females and the con- trols are presented in Additional file 1: Table S7.. - Additionally, the fluorescent signal was detected in some epithelial cells of the reservoir (Fig. - For ChtB4, the fluorescence signal was detected in the spermathecal duct and at the site of attachment of the glandular cells to the duct. - The fluorescence in- tensity of the probes was higher at the attachment site of the duct of the spermathecal reservoir, where the spermathecal gland is located (Fig. - The KSPI transcripts were detected in the spermathecal duct and at the site of attachment of the glandular cells to this duct. - A sum- mary of the fluorescence signals provided by the in situ hybridization for each target gene is shown in Table 2.. - As female mosquitoes mate only once, successful maintenance of the sperm must continue during the female lifespan. - Our gene knockdown analyses revealed insights spe- cific to each of the eight selected transcripts. - Although not all transcripts led to significant loss of the various fitness parameters investigated, their effects were uniquely representative of their role in various aspects of the physiology of the spermathecae.. - Disruption of the trehalose metabolism, such as from a lack of Gld, can severely affect sugar metabolism . - Though we did observe an increase in the length and the area of the eggs following Gld KD, fe- cundity and fertility remained unaffected. - In spite of the. - Two of the transcripts selected are associated with the formation of a chitin layer. - Besides being a major compo- nent of the insect cuticle, chitin also lines the spermathecal reservoir, forming a continuous cuticular layer with the spermathecal duct. - The GALNT6 enzyme is part of the UDP-N- acetylglucosamine (UDP-GlcNAc) pathway, which is in- volved in the maintenance of the exoskeleton of insects [41]. - The assays were performed for each transcript, and staining measurements were taken in different portions of the spermathecae. - Based on the different expres- sion profiles, we reasoned that GALNT6-KD may lead to disruption of the chitin metabolism affecting the sper- mathecal organization in virgin females.. - The high levels of the ex- pression of KSPI (AeSigP-109,183) in the inseminated spermathecae may either be associated with maintaining homeostasis by inhibiting unregulated proteolysis in the spermatheca reservoir that may lead to sperm damage [46] or protecting it from potential pathogens transmit- ted during mating (i.e., venereal transmission).. - Taking into ac- count that Kazal serine proteases are involved in anti- microbial activity (by inhibiting protease activity of pathogens the expression of KSPI is potentially associated with protection of the sperm from pathogens during their journey within the duct.. - This transcript is highly expressed in the inseminated spermathecae and is also present in the content of the reservoir (i.e., in sperm). - Such high level of AeSigP-66,427 in the inseminated spermathecae compared with the reservoir content may also be the result of an additive effect of the sperm. - The lack of the Na + /Ca 2+ exchange caused by the KD of AeSigP-66,427 thus support the idea of an ionic basis for control of sperm motility and lon- gevity . - The impact on the ion exchange homeostasis regarding the spermathecal microenviron- ment was likely due to the KD of AeSigP-66,427 in glan- dular and epithelial cells, as well as the gametes, and is supported by our findings of the in situ hybridization (positive signal) and by the presence of the transcript in the spermathecal content.. - Mosquitoes mate only once, and a single mating event provides enough sperm to fertilize the eggs for the entire reproductive life of the female [20]. - Hence, a better understanding of the mechanisms that promote successful storage of sperm in the spermathecae may unravel potential targets for the reduction of vector populations in the field and. - aegypti, and highlights aspects of the critical balance between spermatheca gene expression regulation, male sperm via- bility, and overall insect fertility. - Knockdown of the Na + /Ca 2 exchanger AeSigP-66,427 in particularly pro- vided strong evidence for the role of this transcript in sperm motility and fecundity. - This study was performed in accordance with the recom- mendations in the Guide for the Care and Use of Labora- tory Animals of the National Institutes of Health. - Total RNA was extracted from each of the two spermathecae pools (1800 per pool) according to the manufacturer’s protocol. - the expression values of the transcripts from the whole body of virgin sugar-fed males and females were deposited in the. - Validation of RNA-seq was performed using RT-PCR to assess the expression profiles of the eight selected transcripts (Additional file 1: Table S5), following the bioinformatics analysis of the spermathecal transcrip- tome. - To tease out gene expres- sion in sperm present in the spermathecae, expression levels of the same selected transcripts were also assessed in the luminal content of the spermathecae from inseminated females. - To confirm spermatheca-specific profile, the expres- sion profile of each of the eight selected transcripts was also assessed in carcasses (i.e., the body without guts and ovaries), midguts, and ovaries from virgin (non-blood fed) and inseminated (blood fed) females. - The selected genes were amplified by PCR reaction using the Taq 2X Master Mix (BioLabs® Inc.) kit, with 5 μM of the designed primers (Additional file 1: Table S5).. - For dsRNA quality analysis, 6 μg of the samples were run in a 1.5%. - After mating/copulation (i.e., day four after emergence), all males were removed from the cages, and KD effects were assessed individually for each of the eight experimental groups (dsRNA-targeted genes). - Effects of dsRNA microinjections on the expression pro- files of spermathecal genes were assessed by using dupli- cate pools of the spermathecae of ten females 2 days after injection, in the case of virgin females, or 2 days after mating, in the case of inseminated females. - The survival analysis of the injected females was done on data collected from three independent replicates with 100 females each. - A schematic design of the phenotypic experiments is shown in Additional file 2: Figure S9.. - Additionally, an assessment of sperm motility was performed to account for effects of the KD in the gam- etes within the spermathecal reservoir. - This part of the experiment was performed in duplicates for each dsRNA and recorded accordingly (Additional files and 10 Movies S1 through S5).. - For probe synthesis, 1 μg of the purified PCR product generated in the purification step of the dsRNA production (as described above for the RNAi experiments) was used, according to the manufac- turer’s protocol. - Histological sections of the spermathecae of Ae.. - Sperm motility inside of the reservoir one day after mating. - Reduced sperm motility inside of the reservoir one day after mating. - No sperm motility inside of the reservoir five days after a blood meal. - This work utilized the computational resources of the NIH HPC Biowulf cluster (http://hpc.nih.gov).. - All authors interpreted the results, read, revised, and approved the final version of the manuscript. - JMR was supported by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases (Vector-Borne Diseases: Biology of Vector Host Relationship, Z01 AI . - Disclaimer: MRO is an employee of the U.S. - Government work as a work prepared by a military service member or employee of the U.S.. - The opinions and assertions expressed herein are those of the author(s) and do not necessarily reflect official policy or position of Uniformed Services University of the Health Sciences, the Department of Defense (DoD), or Departments of the Army, Navy, or Air Force. - MRO is an employee of the U.S. - Government work as a work prepared by a military service member or employee of the U.S. - This study was performed in accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. - The fine structure of the spermathecae and their ducts in the mosquito Aedes aegypti. - The fine structure of the spermatheca in Anopheles aquasalis (Diptera: Culicidae). - Molecular and cellular components of the mating machinery in Anopheles gambiae females. - Transcriptome profiling of the spermatheca identifies genes potentially involved in the long-term sperm storage of ant queens. - Purification and characterization of chitinase from the integument of the Mediterranean flour moth, Ephestia kuehniella (Lepidoptera: Pyralidae) and its antibacterial role. - Regulation of atrophin by both strands of the mir-8 precursor. - Sexual transfer of the steroid hormone 20E induces the postmating switch in Anopheles gambiae. - An ionic basis for a possible mechanism of sperm survival in the spermatheca of the queen honey bee (Apis mellifera L. - yolk protein uptake in the oocyte of the mosquito Aedes aegypti. - An insight into the transcriptome of the digestive tract of the bloodsucking bug, Rhodnius prolixus. - A deep insight into the sialotranscriptome of the gulf coast tick, Amblyomma maculatum. - A deep insight into the sialotranscriptome of the mosquito, Psorophora albipes
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